The present invention relates to a novel kanamycin nucleotidyltransferase with markedly improved thermostability, a selective marker using the same, and a screening method for thermophilic bacteria such as Thermus thermophilus using said selective marker.
Thermophilic bacteria have attracted attention due to the applicability of their proteins in biotechnology. Since the proteins are stable at extremes of pH, they crystallize easily in comparison to non-thermophilic proteins, are easy to handle, and are useful as a good research material in the study of interrelation between enzyme structure and its function. However, when thermophilic protein is expressed in E. coli, its natural conformation may not be reproduced. These proteins, since standard tools of genetic engineering cannot be employed, cannot be expressed in thermophilic bacteria at high temperatures. This prevents an examination of the biological role of these proteins by knocking out genes with functions that cannot be predicted from the sequence data obtained from the genome project, followed by their reintroduction. Thermus thermophilus, which belongs to a Eubacterium, is an attractive organism which can be grown at the highest temperatures (50-82xc2x0 C.) among organisms whose molecular biology is under study.
The sequence analysis of the entire genome of highly thermophilic Thermus thermophilus is currently in progress. Sequence research on the entire genome will soon be completed in Japan (HB8 strain) and Germany (HB27 strain). As with other genome projects, main interest is not in the sequence itself, but has shifted to functional or structural genomics, which is post-sequencing research.
There is a project to organizationally research the structure and biological function of T. thermophilus protein. Therefore, there is a need to rapidly develop genetic engineering tools. The most indispensable tool is an easy-to-use selective marker.
Until now, there were only 2 selective systems that could be used with T. thermophilus. One system was a method where an auxotrophic host was selected via a plasmid into which the corresponding gene was incorporated. However, the auxotrophic marker is inconvenient for routine use. This is because, preparation of the selection medium is troublesome, and growth of cells on the nutritionally restricted plate, is slow even under the optimal growth temperature.
Another system used a kanamycin nucleotidyltransferase (KNT) gene that could be used only at under 60xc2x0 C. T. thermophilus has sensitivity to general antibiotics, however, the only antibiotic resistant marker that can be used with T. thermophilus is a mutant gene of Staphylococcus aureus KNT. However, since this mutant KNT cannot be used at over 60xc2x0 C. as a selective marker, it is far from ideal. At this temperature which is far below optimal growth temperature (70-75xc2x0 C.), cell growth is extremely slow. Thus, we began work to improve the thermostability of KNT.
Many attempts have been made to improve the thermostability of proteins. Most attempts were designed rationally based on an understanding of the protein folding and structure formation. For example, the introduction of disulfide bonds, re-sequencing of packing of hydrophobic cores and substitution with proline were attempted. To investigate whether it was possible to realize thermostability through amino acid substitution, a comparison was made of the sequences of homologous proteins of thermophilic and non-thermophilic bacteria. In contrast, irrational methods have also been applied in order to increase thermostability of proteins. Most of this research involved the induction of random mutations followed by a single screening in place of directed evolution which comprises repeating cycles of inducing mutation, selecting, and amplifying the selected mutant. Only 2 or 3 cases of research reported successful improvement of thermostability through directed evolution.
The present inventors employing a strategy based on directed evolution toward the upper limit of the growth temperature of T. thermophilus, have succeeded in increasing the thermostability of a kanamycin resistant gene product to the upper limit of the growth temperature. The resultant KNT is a convenient selective marker of thermophilic bacteria such as T. thermophilus. 
In other words, the present invention provides the following (1)-(9):
(1) A mutant kanamycin nucleotidyltransferase having one or more point mutations selected from a group consisting of Met57Leu, Ala62Val, Ser94Pro, Ser203Pro, Asp206Val, His207Gln, Ser220Pro, Ile234Val and Thr238Ala as against the protein comprising the amino acid sequence indicated by SEQ ID NO: 1, and having improved thermostability.
(2) A mutant kanamycin nucleotidyltransferase with improved thermostability, wherein it comprises the amino acid sequence indicated by SEQ ID NO: 2.
(3) The kanamycin nucleotidyltransferase according to (1) above, wherein it comprises the amino acid sequence indicated in SEQ ID NO: 3.
(4) A kanamycin nucleotidyltransferase gene encoding the kanamycin nucleotidyltransferase according to any one of (1) to (3) above.
(5) A plasmid comprising the gene according to (4) above.
(6) A transformant comprising the plasmid according to (5) above.
(7) A selective marker for thermophilic bacteria characterized in that it is the gene according to (4) above.
(8) A method for screening thermophilic bacteria wherein the selective marker according to (7) above is used.
(9) The screening method according to (8) above, wherein said thermophilic bacteria is Thermus thermophilus.
In the present invention, the mutant strain with the greatest thermostability has 19 amino acid substitutions when compared with the form prior to mutation. Thermostability was increase by 20xc2x0 C., however, no great change in enzyme activity per se was observed. Most of the altered residues reside on the surface of the protein molecule. Interestingly, 5 substitutions out of 19 were substitutions of the existing residue by proline. The evolved kanamycin-resistant gene product is capable of becoming a selective marker at the optimum growth temperature of T. thermophilus. The development of such convenient genetic engineering tools will promote the post-sequencing research of T. thermophilus.